Currently, the insecticidal gene widely used in the world for biological control of pests is Bt toxin gene of Bacillus thuringiensis (Bt) (such as: Cry1C, Cry1Ab, Cry1B and Cry1F et al.). Bacillus thuringiensis is an insect pathogenic bacterium. The Bt toxin generated by Bacillus thuringiensis has a specific killing effect to many species of agricultural and forestry pests. Since Belgian Plant Genetic Systems first reported the success of transgenic Bt insect-resistant tobacco in 1987 till today, Bt gene has been transferred into main crops in the world, such as: maize, paddy, cotton, tomato, potato and tobacco. According to the statistics of International Service for the Acquisition of Agri-biotech Applications (ISAAA) in 2012, the area of transgenic Bt cotton grown in China has exceeded 3.9 million hectares, accounting for 71.5% of the total area of the cotton grown in China. However, following the application and popularization of transgenic Bt crops, its possible potential hazards in gene escape, change of microbial ecological structure of soil, drug resistance of species and harm to normal immune system have gradually received the attention of the society. The document entitled “Diversity of Rhizospheric Microorganisms and Bacterial Physiological Groups of Transgenic Bt Maize” (Min Wang et al., Chinese Journal of Ecology, 2010(03)) and “Influence of Transgenic Bt Maize on Bacterial Quantity Diversity of Soil” (Ling Liu et al., Journal of Ecology and Rural Environment, 2011(03)) analyzed the bacterial quantity and diversity of the soil in which transgenic Bt maize is grown indoors and outdoors respectively. The results all show significant difference between the transgenic Bt maize group and the blank control group.
The document “Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine” (Vázquez-Padrón et al., Biochem Biophys Res Commun, 2000(01)) disclosed that, in animal experiment, when intrinsic toxic protein of Bt and extrinsic toxic protein of Bt taken in by a mouse reached 10 mg/kg and 100 mg/kg respectively, T cell ANAE positive rate, spleen index and macrophage phagocytosis of the mouse all were inhibited obviously. The more doses are intaken, the more obvious the inhibiting effect will be. This experiment also discovered that when the cumulative coefficient of Bt toxin protein in animal body was greater than 6.24, it might result in injury of liver, kidney and gastrointestinal tract etc. and in liver and kidney, anomalies of cellular swelling and vacuolar degeneration could be observed and glomerular vascular epithelial lesion could be seen. Of course, it can't be excluded that they were caused by immunoreactions. Meanwhile, long-term use of Bt toxin protein at a large dose may also result in significant decrease of total white blood cells (WBC) number and hemoglobin (HGB) content of animals. This also indicates that Bt toxin protein has obvious toxicity of immunosuppression. Therefore, developing substitute biological effectors with Bt toxin bioactivity (such as: anti-idiotype antibody) is a research hotspot in biological pesticide development field.
In 1974, Danish immunologist Jerne firstly proposed the concept of anti-idiotype antibody in his “Immune Network Theory”. Anti-idiotype antibody (hereinafter referred to as “Anti-Id”) refers to the specific antibody generated directed to the idiotype (hereinafter referred to as “Id”) in the variable regions of antibody molecules. Bona, et al. classified Anti-Id into four types (α, β, γ and ε) based on serological reaction between Id and Anti-Id as well as the function of AId. β-type Anti-Id has the effect of “internal image”, i.e.: has antigenic determinant same as (haptin) antigen, so it may have the function and bioactivity of antigen.
Currently, it is universally believed that Anti-Id with an effect similar to target antigen may be obtained by phage display technology through establishment of a phage antibody library, and specific screening. The process of screening specific antibody by phage display technology is called “Panning” and mainly includes four steps: binding, washing, eluting and amplification. Raats et al. adopted anti-cortisol monoclonal antibody envelope as solid-phase antigen for direct screening. Before screening, a same species of negative monoclonal antibody is used to perform negative screening to avoid screening recombinant antibody fragments bound to the constant region of antibody and successfully screen Anti-Id against cortisol. Goletz et al. also employed phage antibody display system and researched and compared the influence of different elution methods on Anti-Id fragment screening results. Of the eventually screened 96 clones, 28 were positive clones with Anti-Id characteristics. So far, no related materials and products specific to substitutable Bt active effector, particularly Anti-Bt toxin type Anti-Id single-chain antibody (hereinafter referred to as “Anti-Id ScFvs”), have been reported.